Genetic Polymorphism rs6505162 in MicroRNA-423 May Not Be Associated with Susceptibility of Breast Cancer: A Systematic Review and Meta-Analysis

Background MicroRNA-423 (miR-423) rs6505162 polymorphism is found to be associated with breast cancer (BC) risk. However, the results were inconsistent. This study meta-analyzed the literature on possible association between rs6505162 polymorphism and BC risk. Methods PubMed, Embase, Google Scholar, and the Chinese National Knowledge Infrastructure (CNKI) databases were systematically searched to identify relevant studies. Meta-analyses were performed to examine the association between rs6505162 polymorphism and BC. Results None of the five genetic models suggested a significant association between rs6505162 polymorphism and BC risk: allelic model, OR 1.02, 95% CI 0.18–1.28, P=0.85; recessive model, OR 0.99, 95% CI 0.72–1.38, P=0.97; dominant model, OR 0.93, 95% CI 0.72–1.21, P=0.60; homozygous model, OR 1.04, 95% CI 0.66–1.65, P=0.87; and heterozygous model, OR 1.07, 95% CI 0.90–1.28, P=0.45. Similar results were obtained in subgroup analyses of Asian, Chinese, and Caucasian patients. Conclusion The available evidence suggests no significant association between rs6505162 polymorphism and BC risk. These conclusions should be verified in large, well-designed studies.


Introduction
Breast cancer (BC) continues to disrupt the lives of millions of women. For many years, BC has consistently ranked among the top cancers in the women, both in terms of incidence and mortality [1]. As we all know, age, menstrual status (early menarche age and delayed menopause), reproduction (late age at first birth), genetic predisposition (higher incidence among close family members and first degree relatives in the breast cancer patients), lifestyle (saturated fat diet, alcohol excessive intake, and obesity), and so on are generally considered to be the causes of BC [2]. However, most causes of BC are not yet clearly understood. Genetic factors have been reported to play an important role in BC development. For instance, mutation in BRCA1 and BRCA2 and low-penetrance common genetic variants were identified as breast cancer risk factors [3]. Recent research studies have shown that one miRNA can potentially affect the expression of many genes to various degrees, and it could participate in the control of numerous metabolic pathways, including cellular growth and differentiation, suggesting that single nucleotide polymorphisms located within miRNAs can have extremely far reaching effects and may affect the development of multiple diseases, including BC [4][5][6][7][8]. ese results indicated that miRNAs may also be risk factors for BC.
miR-423 is located in frequently amplified region of chromosome 17q11.2 and can produce two mature sequences: miR-423-3p and miR-423-5p [6]. Recent studies have shown that rs6505162: C > A, in pre-miR-423 increases risk of familial BC in families with a strong history of BC [7] and SNP rs6505162 in pre-miR-423 affects the mature miR expression, and then miR-423 may play a oncogenic role in breast tumorigenesis [8]. However, results of a recent metaanalysis including only two case-control studies on rs6505162 showed no relationship between rs6505162 polymorphism and BC risk [9]. Given the limited sample size, there is currently no consensus on whether there exists an association between rs6505162 polymorphism and BC risk.
As some new studies published, we conducted this metaanalysis of all relevant literatures to provide comprehensive and reliable insights. To the best of our knowledge, this is the first meta-analysis especially concerning rs6505162 polymorphism and BC risk, and it has the largest sample at present, compared with those published ones.

Search Strategy.
All clinical and experimental casecontrol studies of polymorphisms in the miR-423 gene and BC published through May 15, 2021, were identified through systematic searches in PubMed, Embase, Google Scholar, and the Chinese National Knowledge Infrastructure (CNKI) databases, without language restrictions. e search terms used were: microRNA-423; miR-423; rs6505162; these three terms in combination with polymorphism, polymorphisms, SNP, variant, variants, variation, genotype, genetic, or mutation; and all of the above terms in combination with breast cancer, mammary cancer, or mammary adenocarcinoma. Reference lists in identified articles and reviews were also searched manually to identify additional eligible studies.

Inclusion Criteria.
To be included in our review and meta-analysis, studies had to (1) have a case-control design for assessing the association between rs6505162 polymorphism and BC risk; (2) be accessible as a full-text article and report sufficient data for estimating odds ratios (ORs) with 95% confidence intervals (CIs); (3) report genotype frequencies; and (4) involve humans rather than animal models.

Data Extraction.
Two authors (ZL and LJD) independently extracted the following data from included studies: first author's family name, year of publication, ethnicity, testing methods, NOS score, P value for Hardy-Weinberg equilibrium (HWE) in controls, control source, sample size, matched parameters, and numbers and genotypes of cases and controls. Discrepancies were resolved by consensus. Only those studies that met the predetermined inclusion criteria were included.

Assessment of Methodological Quality.
To assess the quality of the studies included in this analysis, the Newcastle-Ottawa Scale was applied independently by two assessors (ZL and LJD) [10] (Table 1). On the 10-point Newcastle-Ottawa Scale, scores of 5-9 points (stars) are considered to indicate generally high methodological quality, while scores of 0-4 stars are considered to indicate poor quality [11]. Any disagreements about Newcastle-Ottawa scores were resolved by other authors following a comprehensive reassessment. Only high-quality studies were included in the meta-analysis.

Statistical
Analysis. Unadjusted odds ratios (ORs) with 95% confidence intervals (CIs) were used to assess the strength of the association between rs6505162 polymorphism and BC risk based on genotype frequencies in cases and controls. e significance of pooled ORs was determined using the Z test, with P < 0.05 defined as the significance threshold. Meta-analysis was conducted using a fixed-effect model when P > 0.10 for the Q test, indicating lack of heterogeneity among studies; otherwise, a random-effect model was used. All these statistical tests were performed using Review Manager 5.3 (Cochrane Collaboration).
Publication bias was assessed using Begg's funnel plots and Egger's weighted regression in Stata 12.0 (Stata Corp., College Station, TX, USA), with P < 0.05 considered statistically significant. Figure 1 shows a flowchart illustrating the process of searching for and selecting studies. A total of 294 potentially relevant publications were identified. Of these, we excluded 277 studies during initial screening based on review of the titles and abstracts. During analysis of the full text of the remaining articles, two studies were excluded for investigating other miRNAs [12,13], two studies were excluded because they were review articles [14,15], and one study was excluded because it did not report precise genotypes [16].

Description of Studies.
In the end, 12 studies [7,[17][18][19][20][21][22][23][24][25][26][27] were included in this meta-analysis based on our search strategy and inclusion criteria. eir characteristics and genotype distributions are summarized in Tables 1 and 2, respectively. e distribution of genotypes in controls was consistent with Hardy-Weinberg equilibrium (HWE, P > 0.05) in all but one study [25]. e overall quality of the included studies was adequate, and the mean Newcastle-Ottawa score for the included studies was 6.75 (Table 3).

Journal of Oncology
Total score was calculated by adding up the points awarded in each item.

Sensitivity Analysis.
e robustness of the meta-analysis of 12 studies examining a possible association between rs6505162 polymorphism and BC risk was assessed by repeating the meta-analysis after excluding a study [25] in which the P value associated with HWE was less than 0.05. Deleting these data from the meta-analysis did not alter the results obtained using any of the five genetic models, whether for the entire study population or the Asian population.

Discussion
In order to investigate the relationship between rs6505162 polymorphism and BC risk, a few recent meta-analyses [9,[28][29][30] have reported their findings. However, their results were inconsistent. Meta-analysis by Chen et al. [28] with 16 case-control studies included suggested that rs6505162 polymorphism might be associated with a reduced risk of cancers but not with BC risk in subgroup analysis of 5 case-control studies. Meta-analysis by Zhang et al. [29] with 6 case-control studies included suggested that a significantly decreased cancer risk was observed in lung cancer for rs6505162 but not in BC risk. Meta-analysis by Li et al. [30] with 8 case-control studies included suggested rs6505162 decreases the risk of cancer, showing that it is the protective factor of cancer. But subgroup analysis for BC risk was not performed.
ose previous meta-analyses did not specially focus on BC, much less on BC by subgroup analysis by ethnicity. In order to evaluate available evidence on the possible association between rs6505162 polymorphism in miR-423 promoter and BC risk, a more detailed meta-analysis was performed. Results showed that miR-423 rs6505162 might not be associated with BC risk, regardless of ethnicity. Even though our results supported previous studies [28,29], given larger sample with 12 case-control studies included, ours should be more convincing.
Although null results were obtained in the current study on rs6505162 polymorphism with larger sample, we really hope they would provide a reference for future studies. Nonetheless, the work still has several limitations that may affect interpretation of the results. Firstly, the P value for HWE in one study [25] was less than 0.05, making these study populations not being representative of the broader target population. Nevertheless, sensitivity analyses showed that deleting the study did not alter the results. Secondly, the studies may be subject to performance bias, attrition bias, and reporting bias, although Newcastle-Ottawa scores were more than 5 for all studies, indicating high quality. irdly, additional confounding factors such as age, gender, and tumor status may affect the results. In order to reduce the effect of those confounding factors above on the results, we have tried our best to make stratified analysis based on those factors. In the end, only gender could be taken into account. A total of 5 case-control studies [18][19][20][21]27] of which the patients were all definitely female were selected to investigate the relationship between rs6505162 polymorphism and BC risk on females. Nevertheless, these studies either did not report age and tumor status or aggregated them in different ways, resulting in a failure to include them in the metaanalysis. Lastly, methods used to test for polymorphisms were not uniform and they varied in sensitivity and specificity, which may reduce the robustness of the meta-analysis.    In conclusion, this study performed an extensive assessment based on a larger sample size than the previous pooled analysis and suggested no significant association between miR-423 rs6505162 polymorphism and BC risk. ese conclusions should be verified in large, well-designed studies.

miR:
MicroRNA BC: Breast cancer HWE: Hardy-Weinberg equilibrium CNKI: Chinese National Knowledge Infrastructure OR: Odds ratios 95% CI: 95% confidence interval TNBC: Triple-negative breast cancer HB: Hospital-based source of control PB: Population-based source of control PCR: Polymerase chain reaction RFLP: Restriction fragment length polymorphism HRM: High-resolution melting ARMS: Amplification refractory mutation system.

Data Availability
e data used to support the findings of this study are included within the article.

Conflicts of Interest
e authors declare that they have no conflicts of interest.